JPS59175888A - Gene resistant to novobiocin and plasmid - Google Patents

Abstract

PURPOSE:To succeed in preparation of plasmid having genetic index of novobiocin resistance from DNA of chromosome of Streptomyces spheroides in a system using plasmid derived from ray fungus. CONSTITUTION:A gene resistant to novobiocin contained in DNA corresponding to a fragment having 4.1 megadalton obtained by scissoring DNA of Streptomyces spheroides with restricted enzyme BclI, having a scission map of restricted enzyme shown by the figure. This gene is integrated into a vector plasmid to give hybrid plasmid.

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention The present invention relates to an antibiotic resistance gene of actinomycetes and a hybrid cilasmid incorporating the same.

Actinobacteria are the most important fermentation microorganisms that are widely used in the microbial industry as producers of useful substances such as antibiotics.

If recombinant DNA technology can be used to breed actinomycetes or elucidate their genetic properties, we can expect extremely valuable industrial benefits.

Summary of the Invention The present inventors have developed a DNA containing a novobiocin resistance gene from Streptomyces sphaeroides chromosomal DNA using a system using Streptomyces sphaeroides as a host and cilasmid derived from Streptomyces.
We succeeded in isolating the fragment and constructing a plasmid with the genetic indicator of novobiocin resistance.

The present invention is based on the research results of the present inventors.

Therefore, the novobiocin resistance gene according to the present invention has the following characteristics.

(b) This gene is derived from Streptomyces sphaeroides.
It is contained in the DNA corresponding to the 4° 1 megadalton fragment obtained by cleaving the DNA of s) with the restriction enzyme Bcl].

(b) The restriction enzyme cleavage map of this gene is shown in Figure 1.

In addition, 4 with the novobiocin resistance gene according to the present invention
, 1 megadalton DNA fragment was obtained by cleaving the DNA of Streptomyces sphaeroides or its mutant strain with the restriction enzyme Bcl I.

Furthermore, the hybrid louse P according to the present invention is
The novobiocin resistance gene having the following characteristics is incorporated into a vector cilasmid.

(b) This gene is derived from Streptomyces sphaeroides (Streptomyces 5 pheroicl).
It is contained in the DNA corresponding to the 4.1 megadalton fragment obtained by cleaving the DNA of es) with the restriction enzyme Bc11.

(b) The restriction enzyme cleavage map of this gene is shown in Figure 1.

Shirasumi P having the nosebiocin resistance marker according to the present invention can be particularly used as a vector for recombinant DNA experiments using actinomycetes, which are producing bacteria of useful substances such as antibiotics, as a host. Extremely valuable results can be expected by breeding or genetic analysis of actinomycetes through recombinant DNA experiments using such an actinomycete host-vector system.

The novobiocin (hereinafter referred to as NB) resistance gene according to the present invention is contained in a DNA corresponding to a 4.1 megadalton fragment obtained by cutting Streptomyces suide DNA with the restriction enzyme BcIT,
The restriction enzyme cleavage map is as shown in FIG.

Here, the DNA of S. sphaeroides is digested with restriction enzyme Bc.
The DNA j corresponding to the 4.1 megadalton fragment obtained by cutting with I means not only this Bcl I cleavage fragment itself but also DNA having the same base sequence as the Bcl I cleavage fragment. A specific example of the latter DNA is S.

A Bal I cleavage fragment of a mutant strain of Sphaeroides and a synthetic NA.

Preferred specific examples of the NB resistance gene according to the present invention include S,
The DNA of Sphaeroides or its mutant strain is digested with restriction enzyme B.
4.1 megadalton (M
This is included in the fragment d). The present invention also provides this 4. It also concerns fragments of IMd.

Gene Production The NB resistance gene according to the present invention is usually obtained from S. sphaeroides or mutant strains thereof, which may be artificially synthesized.

(1) The NB producing bacterium used as the producing bacterium DNA donor is Streptomyces sphaeroides or a mutant strain thereof.

3. As spheroides, S, spheroides AT
There is CC23965. This bacterium has been deposited as ATCC 23965 at the American Type Culture Collection in Maryland, USA.

In view of the nature of the present invention, the mutant strain of S. sphaeroides is D, which gives the restriction enzyme cleavage map shown in FIG.
It should have NA.

(2) Gene cloning Total DNA was extracted from the cells of the selected NB-producing bacteria using the 5DS-phenol method (M, D, Smlth et al: t4t
Mds in Enzymo 1 ogy, 12.545
(1967)). When the extracted DNA is digested with restriction enzyme + (cl I), a DNA fragment containing the desired gene is obtained together with various DNA fragments.

In order to extract the desired gene-containing fragment from the DNA fragment mixture obtained in this manner and to obtain it in large quantities, it is necessary to incorporate it into an appropriate viral or circular plasmid as a vector and to transform the host bacterium by generating a hybrid plasmid. ffi gene recombination techniques can be utilized, including transformation, transduction, or transfection. Regarding general genetic recombination technology, for example,
You can refer to "Gene Manipulation Experimental Methods" (Kodansha), edited by Yasutaka Takagi.

An example of such a recombinant technique is as follows. That is, when the donor DNA is cleaved with Bcl I, the vector plasmid is allowed to coexist and this vector plasmid is also simultaneously cleaved at the Rcl 1 site, or this vector plasmid is separately cleaved with Bal.
After cutting the donor DNA with Bcl
The vector is obtained by mixing with the I cut product and treating with a suitable gauze, such as T4 ligase. As a vector plasmid that can be used in this case, one that has an f(ell cleavage site and gives a propagable fragment when cut with 13all) is generally suitable.The vector plasmid is derived from actinomycetes, Escherichia coli, etc. , Bacillus subtilis and Sendai, but to name a few specific examples, psF' 689, psF619 and pSF 7 are derived from actinobacteria.
65 (for the above, refer to Japanese Patent Application Laid-Open No. 188600/1983).

Also, please refer to the same publication for the mycological properties of each host bacterium), and others. To obtain a target plasmid from a host bacterium, the technique described in the above-mentioned publication and other techniques commonly used for genetic recombination may be followed.

Representative of these vector plasmids is the 4.8 Md psF 765 derived from Streptomyces.

Its host bacterium, Streptomyces fradiae S
F 765 (Feikoken Article No. 124) (and the mycological properties of this bacterium) was published in JP-A-57-18860 as mentioned above.
It is listed in Publication No. 0 and is ready for sale.

Another representative example of vector cilasmid derived from actinomycetes is 9. IMd's psF 689. The host bacterium S. platensis 5F689 (Feikoken Jokyo No. 121) (and the mycological properties of this bacterium) are disclosed in JP-A No. 57-188600 and JP-B No. 45-6076, as mentioned above.
It is listed in the publication and is ready for sale.

Next, host bacteria are transformed with the obtained plasmid mixture. As a host bacterium, an appropriate host bacterium can be selected from among actinomycetes, Escherichia coli, Bacillus subtilis, and other microorganisms, especially those with high transformation efficiency and high sensitivity to NB, depending on the type of four vectors used. good. When the vector is an actinomycete plasmid, an actinobacterium is used as the host bacterium, and specific examples in this case include S. Lividans 66 (Feikoken Bacteria No. 6982).

In order to introduce a vector incorporating a DNA fragment of an NB-producing bacterium into a host bacterium, the most efficient method is selected depending on the host bacterium and the type of vector. The most common method is to transform bacterial zorodoplasts.

For selection of recombinants obtained by transformation, genetic indicators possessed by the vector used, such as antibiotic resistance, pock formation (M, , ■, Bibb et al: M
o1ec, gen, Crenet, 154.155
(1977), Nature 274.398 (1978
)) etc. are available.

In order to select those containing the NB resistance gene from the recombinants thus obtained, it is most efficient to select NB-resistant strains produced by the expression of NB resistance. To do this, first, the minimum inhibitory concentration of NB against the host bacteria is determined, and clones that grow on a selective medium containing NB at or above that concentration are selected.

The obtained NB-resistant clones were cultured, and the bacterial cells were cultured using a known method [Har+1Ien et al: J, Bact.
erlol, 135.227.19781 and inserted DNA by restriction enzyme treatment.
The fragments can be removed and the DNA fragment containing the NB resistance gene isolated by agarose gel electrophoresis. In addition, confirmation that it is a clone of the NB resistance gene may be performed by reintroducing the isolated plasmid into the host bacterium and examining the expression of NB resistance.

By further cutting the obtained DNA fragments with various restriction enzymes, performing agarose gel electrophoresis and analyzing the size of each fragment, a restriction enzyme cleavage map can be created, and each fragment can be further cut into an appropriate vector. By integrating the gene into the NB and recloning, it is possible to perform structural analysis of the NB chuji resistance gene and clarify its lLY nature. As described above, the restriction enzyme cleavage map of the DNA carrying the NB resistance gene of the present invention is as shown in FIG.

Hybrid cilasmid The hybrid cilasmid according to the present invention contains the NB resistance gene described above.

A specific example of the hybrid plasmid of the present invention in which the vector plasmid into which the NB resistance gene is to be incorporated is an actinomycete plasmid is as described above in connection with cloning.

One specific example of a hybrid plasmid according to the invention is pMs32.

pMs 32 is Streptomyces cilasmid psF 765 (4
, 8Md) containing the NB resistance gene 4. IMd's DN
Actinomycete plasmid (8,9Md) formed by ligating A fragments
(Figure 2). This plasmid has been deposited as a bacterium transformed with it at the Microtech Institute (details will be described later).

When each of these NB-resistant plasmids is transformed into a corresponding host bacterium, the host bacterium can express NB resistance. For example, when Streptomyces lividans 66 is transformed with cilasmid I) MS 32 (Fig. 2) in which a DNA fragment containing the NB resistance gene of the present invention has been incorporated into Streptomyces cilasmid pSF 765, 50μ
A recombinant showing NB resistance of g/ml was obtained.

Experimental Examples Example 1 [Preparation method of pMS32 and) Properties of I biocin resistance gene] Streptomyces sphaeroides ATCC2396
5 was inoculated into 80 ml of MYG (1% malt extract, 0.4% yeast extract, 0.5 tiglucose, pH 7.0) and cultured with shaking at 28'C for 2 days.
Bacteria were collected by centrifugation at 00 x g for 710 minutes. From this bacterial cell, the known method of Smith et al.
n Enzy-mologg, 12.545.19
Total DNA was extracted and purified using TESL buffer (10mM Tris-HCI, pH 8,0,1mM
Dialyzed against EDTA, 2.5 mM NaCl),
This was used as donor DNA.

6 μg of the donor DNA thus obtained and 7651.5 μg of plasmid psF were mixed, and the total amount was 100 μg.
ml of 10mM Tris-HCI XpH7,4,6
mM dithiothreitol, 10 mM MgC12,5
Restriction enzyme BCI I in a reaction solution with 0mM NaCl composition.
5 units were added and reacted at 50"C for 2 hours. TESH buffer (0.2M Tris-HCI, p
H8, 0, 20mM dithiothreitol, 50mM
100 μm of a phenol solution saturated with NaC1) was added and shaken for 1 minute, and after centrifugation at 10,000×g/5 minutes, the upper layer (aqueous layer) was removed with a Pasteur pipette. This aqueous layer containing DNA was subjected to ethyl ether extraction to remove phenol, then 300 μm of ethanol was added, left at -80°C for 2 hours, and then extracted with 10,000 μm of ethanol.
DNA was precipitated by centrifugation at 0 × g, /5 min.

The DNA thus obtained was washed with 500 μl of ethanol, dried under reduced pressure, and then dissolved in 40 μm heat-sterilized pure water. This DNA solution was heat-treated at 70°C for 10 minutes, then gradually cooled to room temperature, and a buffer solution (0,6
6M Tris-HCI, pH 7, 6, 66mM MgC
12,0,1M dithiothreitol, 10mM AT
P) Add 5 μl and 6 μm (1 unit) of T4 ligase and react at 22”C for 2 hours to obtain recombinant corpse D of pSF 765 and Streptomyces sphaeroides DNA.
NA was prepared. The host bacterium Streptomyces lividans 66 (Lomovskaya e.
tal:Genetics, 68.341.1971
) can be transformed using the known method of Chater et al.
r, Topics Mlcrobiol.

Immunol, 96.69.1981). That is, 80 ml of YEME medium (
34% sucrose, 0.3% yeast extract, 0.5% bactopezotone, o, 3% malt extract, 1% glucose,
5 mM MgCl2.0.5 tiglycine, pH 7.0
) was inoculated with Streptomyces lividans 66 and 3
Culture with shaking at 2°C for 36 hours, 10,000
The hyphae were collected by centrifugation at 10 g and washed once with 10 tish sugar solution, followed by 10 ml of P medium (0.3 M sucrose, 1.
4mM K2SO4, 10mM MgC12,0,4m
M KH2PO4,25 sea bream CaCl2.5 sea bream TES buffer, pH 7,2, 11500 volume I trace element solution). Next, egg white lysozyme was added to the final concentration of 1 mg/ml, and the mixture was kept at 32°C for ω minutes to form protoplasts, and the mycelia that did not form into protoplasts were removed using a cotton sieve. Protoplasts were collected by centrifugation at 800×g/7 min, washed once with P medium and then suspended in 2 ml P medium (composition of trace element solution (in 1 liter): 40 mg ZnCl2.
200 mg FeCl2” 6H20, 10 mg Cu
C12” 2 H2O, 10mg MnC12・4H
2O, 10mg Na2B4O7 @ 10 H2O, 10
mg (NH4)6Mo7024.4H2O).

Protoplast solution 100 μm, P prepared to 3/2 concentration
-Maleic acid buffer (2.5% sucrose, 1.4mM K2
SO4, 11500it trace element solution, 100mM
CaCl2.50mM Trim-maleic acid, p
H8,0) 100μ! .

50 μl of recombinant DNA solution and 375 μl of polyethylene glycol solution (10 μl of polyethylene glycol solution)
0033%,/P-maleic acid buffer), left at room temperature for 1 minute, diluted with 5 ml of P medium, and diluted with 800%
Protoplasts were collected by centrifugation at X g for 710 minutes and suspended in 2 ml of P medium. R2YB agar medium (0.3M sucrose, 1.4mM K2SO4, 50mM MgCl2
．． 1% glucose, 0.01 ticazamino acid, 17500
Trace element solution, 0.4 mM K)12PO4,20
mM CaCl2.0.3 Proline, 25 mM T
ES buffer, pH 7, 2, 5mM NaOH 10, 5%
yeast extract and 2.2 agar) and cultured at 31°C for 4 days. Bacteria on agar medium regenerated from protoplasts were transferred to Pennet medium containing nosepiocin or μg/fnl (1 tiglucose, 0.2% - (futon, 0.1% meat extract, 0.1 t yeast extract, 2% Agar, pH 7.0) was replicated using a velvet cloth and cultured for 2 days at 31°C.As a result, 5 knobbiocin-resistant clones of the linea were obtained, and these colonies on the selective medium were cultured. Scrape each with Aze, and using a glass Botter homogenizer, add sterilized water to 0.1 to 0.
．． It was dispersed in 2 ml, spread on R2YE medium, and cultured at 31°C for 5 days.

5 stocks obtained)? Biocin-resistant Strephtomyces lividans 66 was inoculated into 80 ml of YEME medium, and the cultured cells were shaken at 31°C for 2 days.
35.222.1978). When Streptomyces lividans 66 was transformed again with these plasmids, notehyosine resistance was expressed in all cases. When the plasmids obtained from the 5 strains were cut with the restriction enzyme Bet l and examined by 1% agarose gel electrophoresis, in addition to psF 765 of 4.8 Md, 4. A common NA fragment of I Md was observed. Furthermore, 5ell of these plasmids.

After detailed examination by cutting with restriction enzymes such as Ava I and Pvu II, we found that 4. The direction in which the I Md DNA fragment was integrated into plasmid psF765 was not constant. However, 4. Streptomyces lividans 660 transformed with two types of plasmids with different insertion directions of I Md DNA were all resistant to nosebiocin, and there was no change in the resistance due to the difference in the insertion direction. . 4. ■MdD
The nosebiocin resistance gene encoded by NA is designated as the 72 biocin resistance gene. Figure 1 shows the restriction enzyme map of the DNA fragment, and psF 765 incorporating this DNA fragment is named pMs32, and Figure 2 shows its restriction enzyme map. showed that.

1) Accession number ATCC23965 2) Mycological properties (1) Spore morphology The tips of the bifurcated branches are spiral-shaped.

Mature spore chains are generally long, with 50 or more spores per chain. This form is found on yeast malt agar, oatmeal agar, starch mineral salt agar, and glycerol asnoline agar. The surface of the spore is smooth.

(2) Colony color: Aerial mycelia appear yellow on yeast malt agar, oatmilk agar, starch inorganic salt, and glycerol-asparagine agar.

(3) Color of the back side of the colony: The back side of the colony does not exhibit a characteristic color on yeast malt agar, oatmeal agar, starch inorganic salt agar, glycerol, and asparagine agar (colorless or pale yellow to light yellowish brown).

(4) Color in the medium: Melanin pigments are not formed in pezotone yeast agar, tyrosine agar or tryptone-yeast broth.

Other pigments also do not form on yeast malt agar, oatmeal agar, starch mineral salt agar, or glycerol, asparagine agar.

(5) Sugar utilization: D-glucose, L-arabinose, sucrose, D-xylose, D-mannitol, D-fructose, and rhamnose are utilized. i-inositol and raffinose are hardly used.

S. fradiae SF 765 1) Accession number: Microtechnical Research Institute No. 124 (May 1, 1929) 2) Mycological properties, see JP-A No. 188600/1983 S. platensis SF 689 1) Accession number: Microtechnical Research Institute No. 121 (May 1, every other year of the Showa era) 2) Mycological properties JP-A-57-188600 and JP-A-Sho 45-6
See Publication No. 076.

S. Lividans 66 1) Accession number: Microtechnical Research Institute Bacteria No. 6982 (March 10, 1920) 2) Mycological properties (1) Forms a spiral spore chain, and the mature spore chain is medium-sized. Long, - consisting of 10 or more spores per chain. This form is compatible with yeast malt agar, oatmilk agar, starch inorganic agar and glycerol agar.
The spore surface is smooth and can be seen on line agar.

(2) Aerial mycelium exhibits a gray to red color on yeast malt agar, oatmill agar, starch inorganic salt agar, and glycerol-asparagine agar.

(3) The color of the back side of the bacterial cells ranges from the characteristic pale to dark Daleitspurple or Daleitsschreissno R-purple on yeast malt agar, oatmeal agar, inorganic salt agar, and glycerol-asparagine agar. show. The pigment on the back side of this bacterial cell is 0.0
By adding 5N caustic soda, the color changes from reddish or iolet to blue. Moreover, violet or blue changes to red by addition of 0.05N hydrochloric acid.

(4) Soluble pigments: Melanin-like pigments are not formed in peptone-yeast iron agar, tyrosine agar, or tryptone yeast liquid media. Blue or wild iolet pigments are observed in yeast malt agar, oatmeal agar, starch inorganic salt agar, and glycerol-asparagine agar. This pigment is sensitive to pH and changes in the same way as described for the pigment on the back side of the bacterial cell.

(5) Sugar assimilation = D-glucose, L-arabinose, D-xylose, l-inositol, D--r 7
= )-ru, D-fructose, rhamnose, sucrose and raffinose are all assimilated. however,
Idle growth on sucrose and raffinose is poor compared to other sugars.

S. Lividans 66 (pMs 32) 1) Accession No. 6984 (March 10, 1984) 2) Mycological properties Other than the properties introduced by cilasmid pMs 32,
S, IJ Bidans 66 (Feikoken Bibori No. 6982)
It is the same as that of

[Brief explanation of the drawing]

FIG. 1 shows a restriction enzyme cleavage map of the nobiocin resistance gene according to the present invention. FIG. 2 shows the restriction enzyme cleavage map of plasmid pMs 32 according to the invention. Applicant's agent Kiyohiro Inomata 1 52 Figure sg+n Procedural amendment 1986-10) Commissioner of the Patent Office Kazuo Wakasugi 1, Indication of case Patent Application No. 52276 of 1982 2, Title of invention Nobiocin resistance gene and cilasmid 3, relationship with the case of the person making the amendment Patent applicant (609) Meiji Seika Co., Ltd. 7, "Detailed description of the invention" column 8 of the specification subject to the amendment, Contents of the amendment (1) "Dithiothreitol" on page 14, line 14 of the specification is corrected to l'-EDTAJ. (2) On the 4th line from the bottom of page 18, correct [5cllJ to (-8allJ). (2)

Claims (1)

[Claims] 1. A Noto biocin resistance gene having the following characteristics. (b) This gene is derived from Streptomyces sphaeroides.
It is contained in the DNA corresponding to the 4.1 megadalton fragment obtained by cutting the DNA of es ) with the restriction enzyme Bel I. (b) The restriction enzyme cleavage map of this gene is shown in Figure 1. 2. The nosebiocin resistance gene according to claim 1, wherein the DNA containing the gene is derived from Streptomyces sphaeroides or a mutant strain thereof. 3. A 1 megadalton DNA fragment containing a nosebiocin resistance gene obtained by cleaving the DNA of Streptomyces sphaeroides or its mutant strain with the restriction enzyme Bel I. 4. A hybrid plasmid obtained by incorporating a nosebiocin resistance gene having the following characteristics into vector plasmid P. (b) This gene is derived from Streptomyces 5 pheroides.
It is contained in the DNA corresponding to the 4.1 megadalton fragment obtained by cleaving the DNA of es) with the restriction enzyme Bcl I. (b) The restriction enzyme cleavage map of this gene is shown in Figure 1. 5. The hybrid plasmid according to claim 4, wherein the vector plasmid is an actinomycete plasmid. 6. The NoI biocin resistance gene is present in Streptomyces
The DNA of Sphaeroides or its mutant strain is digested with restriction enzyme B.
4.1 megadalton DN obtained by cutting with cl I
Claims 4 to 5 incorporated in the form of A fragment
The hybrid cilasmid according to any one of paragraphs.